Myeloproliferative disorders (MPD) are clonal disorders of haematopoietic progenitors, and include the classical MPD chronic myeloid leukaemia (CML), polycythaemia vera (PV), essential thrombocythaemia (ED and primary myelofibrosis (PMF), as well as chronic eosinophilic leukaemia (CEL), chronic myelomonocytic leukaemia (CMML), and systemic mastocytosis (SM) and others. In the past two decades, mutant alleles have been identified in CML, CMML, CEL and SM2-5; and in each case the causative mutation results in constitutive activation of tyrosine kinase signalling. The genetic causes of the most common MPD remained unknown until the identification of mutations that activate Janus kinase 2 (JAK2) signalling in most patients with PV, ET or PMF(1, 2, 3, 4). JAK2 is a member of the Janus family of cytoplasmic non-receptor tyrosine kinases, which also includes JAK1, JAK3 and TYK2. The mutation is a guanine-to-thymidine substitution at position 1849 in the JAK2 coding sequence (GenBank accession no. NM_004972, SEQ ID NO:1), numbering starting at the ATG start codon, corresponding to position 2343 of SEQ ID NO: 1. Such a mutation results in a substitution of valine for phenylalanine at amino acid 617 of the JAK2 protein (JAK2V617F), within the JH2 pseudokinase domain (5). Loss of JAK2 autoinhibition results in constitutive activation of the kinase, analogous to other mutations in MPDs and leukemia that aberrantly activate tyrosine kinases (6,7,8).
Direct sequencing is only sensitive down to about 20% of mutant DNA in a wild-type background (9, 10). This issue is quite relevant to chronic myeloid disorders, where blood and marrow are often composed of a mixture of neoplastic and residual normal hematopoietic elements. This is especially the case of ET and MDS, in which phenotypically apparent gene mutations may be present in tiny clones comprising less than 10% of the total marrow cell population. James et al. (11) explored this issue specifically with respect to JAK2 1849 G-T by performing a series of mixing experiments with HEL erythroleukemia cells, which bear the JAK2 mutation, admixed with TF-1 erythroleukemia cells, which do not. They failed to detect the mutated allele when it was present in <5% of the total DNA. With homozygous mutant patient DNA diluted in DNA from a healthy person, sequencing was even less sensitive (10%) than it was with the cell lines (12).
A common method used for the detection of nucleic acid mutations is the Amplification Refractory Mutation System (ARMS). It exploits the fact that oligonucleotide primers must be perfectly annealed at their 3′ ends for a DNA polymerase to extend these primers during PCR (12). By designing oligonucleotide primers that match only a specific DNA point mutation, such as that encoding JAK2 V617F, ARMS can distinguish between polymorphic alleles. Therefore, these techniques go by the alternative names of “allele-specific PCR” (AS-PCR) or “sequence-specific primer PCR.” The ARMS sensitivity is up to 1 to 2% (13) mutant DNA in a wild-type background.
Real-time monitoring of PCR product accumulation during thermocycling can be of value as a semiquantitative method and DNA-melting curve assays can be used in conjunction with real-time PCR. Likewise, James et al. (14) compared fluorescent dye chemistry sequencing with two different real-time PCR based mutation detection systems, one using a LightCycler instrument (Roche Diagnostics) and the other using a Taqman ABI Prism 7500 machine (Applied Biosystems). These real-time PCR techniques detected 0.5 to 1% of HEL cell line DNA diluted in TF-1 cell line DNA and 2 to 4% of homozygously mutated patient DNA diluted in DNA from a healthy person.
A Restriction Fragment Length Polymorphism (RFLP) analysis is possible since the JAK2 1849 G-T mutation abolishes a motif in the wild-type JAK2 sequence that is recognized by the restriction enzyme BsaXI. Although abolition of a restriction site is not as satisfying as creation of a new site, because a negative enzymatic cleavage reaction could be due either to absence of the mutation or to failure of the digestion procedure, it can be useful as a first pass analysis. Reported proportional sensitivity depends in part on the method used to detect the fragments and is approximately 20% mutant DNA in Wild-type background (15, 16).
Pyrosequencing is a method of rapid genotyping that depends on the liberation of pyrophosphate (PPi) whenever a dNTP is incorporated into a growing DNA chain during template-driven DNA polymerization (17). Pyrosequencing of JAK2 using the automated PSQ HS 96 system (Biotage, Uppsala, Sweden) has been attempted by several groups (17, 18) with dilution experiments similar to those described above showing a reported assay sensitivity of 5 to 10% mutant allele in a wild-type background.
Several other mutation detection techniques have been described, including single stranded conformational polymorphism (SSCP) analysis, denaturing gradient gel electrophoresis (DGGE), denaturing high-performance liquid chromatography (DHPLC), single-nucleotide primer extension assays (Pronto), and others. In fact, DHPLC can detect the genomic DNA mutation underlying JAK2 V617F reliably, and it can detect mutations at a proportionality of <1 to 2%. However, DHPLC and the other techniques are either technically challenging or labor-intensive or both. They either do not allow high throughput at a cost suitable for a clinical laboratory (SSCP and DGGE) or require a considerable initial investment for equipment (DHPLC).
Theoretically, protein-based techniques could also be used to detect the JAK2 V617F mutation, but these are generally cumbersome, and access to such resources is limited. Therefore, protein-based assays are usually not preferred if DNA- or RNA-based tests are feasible.
European patent application EP1692281A discloses a method for the detection of G1849T JAK2 mutation based on PCR amplification.
The methods of the prior art show several limitations. First of all the lower level of sensitivity, that allows detection of the mutant JAK2 sequence down to 1% of the sample in the best cases. Such a sensitivity requires the enrichment of the mutants via granulocytes-isolation before extraction. This is a time consuming and labor-intensive step resulting in about 2 additional hours to the already long procedures (from 2 to 5 hours) requested for the diagnosis. Furthermore, all the methods previously illustrated are relatively labor intensive and expensive, often requiring specialized equipment that may not always be readily available.